Vitamin A derivative all-trans-retinoic acid regulates the expression of over 530 different genes. Consequently, the levels of retinoic acid during embryogenesis are controlled in a spatially and temporally precise manner. However, the molecular mechanisms underlying this regulation are not yet fully understood. The long-term objective of this project is to determine the role of short-chain dehydrogenases/reductases (SDRs) in the regulation of retinoic acid biosynthesis in health and disease. Recently, we have identified a new member of the SDR superfamily of proteins in frogs, rdhe 2, that is highly active as an all-trans-retinol dehydrogenase and is critical for embryonic development in Xenopus laevis. Importantly, there appears to be a functional equivalent of the frog rdhe2 in mammals, which exhibits an all-trans-retinol dehydrogenase activity and is expressed during early embryonic development. We propose that this novel enzyme, named RDH-E2S, is essential for retinoic acid biosynthesis in mammals during embryogenesis and, possibly, in adulthood. To test this hypothesis, we will characterize the catalytic properties of mammalian RDH-E2S and determine its contribution to retinoic acid biosynthesis in vivo using genetically modified mouse model (Specific Aim 1). Our preliminary studies indicate that silencing of retina short-chain dehydrogenase/reductase 1 (retSDR1) gene expression in human cells results in significant increase in the levels of both retinoic acid and retinaldehyde, which translates into dramatic upregulation of retinoic acid-responsive genes. This finding suggests that the rate of retinoic acid biosynthesis is determined by the relative activities of retinol dehydrogenases and retinaldehyde reductases, which together control the levels of retinoic acid precursor, retinaldehyde. To test this hypothesis, we propose to characterize the catalytic properties of retSDR1 and to determine its role in the regulation of retinoic acid levels in vivo using genetically modified mice, human skin organ culture, and Xenopus laevis in vitro model of early embryonic development (Specific Aim 2). These studies will fill the gaps in our understanding of the mechanisms responsible for the maintenance of retinoic acid homeostasis by providing new information regarding the roles of potentially important components of the retinoid regulatory system. The results of these studies will be important for understanding the pathophysiology of disorders associated with disruptions of retinoid homeostasis, such as fetal alcohol syndrome, alcoholic liver disease, carcinogenesis, and diabetes.